The sporulation and germination-outgrowth developmental cycle of the bacterium Bacillus subtilis provides a model biological system for examining intracellular differentiation. This project has the following objectives, all of which are primarily concerned with the regulation of gene expression. (1) An examination of gene regulation during spore outgrowth and spore formation. We will continue our study of the control of several enzymes (trehalase and threonine deaminase) which are synthesized at unique periods during spore outgrowth. We will also study the control of a sporulation specific enzyme, dipicolinic acid synthetase. Both systems will be studied in vitro using gene isolation methods and the RNA polymerase activities isolated from various developmental periods, forespores and spore mother cells. 2. We will continue our study of the chromosomal organization and expression of transfer RNA genes during spore development, using as probes, plasmids (pBR313) in which we have previously cloned tRNA and rRNA genes of B. subtilis. (3) We will undertake the construction of a temperature sensitive suppressor gene which can be used to identify and regulate the level of sporulation specific gene products. This system will also be used to examine for preferential translation in the forespore and in spore mother cell. (4) We will examine whether chromosome re-arrangements occur during the developmental cycle. This will be carried out by hybirdization of radioactive DNA or RNA probes of genetically known gene segments to DNA restriction fragments which hve been separated by a two dimensional gel electrophoresis method. A model system will first be tested in trpE26 strains of B. subtilis which are known to have chromosomal translocations.